title: De novo transcriptome sequencing of axolotl blastema for identification of differentially expressed genes during limb regeneration.
abstract: BACKGROUND
  Salamanders are unique among vertebrates in their ability to completely regenerate amputated limbs through the mediation of blastema cells located at the stump ends. This regeneration is nerve-dependent because blastema formation and regeneration does not occur after limb denervation. To obtain the genomic information of blastema tissues, de novo transcriptomes from both blastema tissues and denervated stump ends of Ambystoma mexicanum (axolotls) 14 days post-amputation were sequenced and compared using Solexa DNA sequencing.
  
  
  RESULTS
  The sequencing done for this study produced 40,688,892 reads that were assembled into 307,345 transcribed sequences. The N50 of transcribed sequence length was 562 bases. A similarity search with known proteins identified 39,200 different genes to be expressed during limb regeneration with a cut-off E-value exceeding 10-5. We annotated assembled sequences by using gene descriptions, gene ontology, and clusters of orthologous group terms. Targeted searches using these annotations showed that the majority of the genes were in the categories of essential metabolic pathways, transcription factors and conserved signaling pathways, and novel candidate genes for regenerative processes. We discovered and confirmed numerous sequences of the candidate genes by using quantitative polymerase chain reaction and in situ hybridization.
  
  
  CONCLUSIONS
  The results of this study demonstrate that de novo transcriptome sequencing allows gene expression analysis in a species lacking genome information and provides the most comprehensive mRNA sequence resources for axolotls. The characterization of the axolotl transcriptome can help elucidate the molecular mechanisms underlying blastema formation during limb regeneration.
authors.affiliation: Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan.
authors.forename: Cheng-Han
authors.initials: CH
authors.lastname: Wu
authors.forename: Mong-Hsun
authors.initials: MH
authors.lastname: Tsai
authors.forename: Chia-Chuan
authors.initials: CC
authors.lastname: Ho
authors.forename: Chien-Yu
authors.initials: CY
authors.lastname: Chen
authors.forename: Hsuan-Shu
authors.initials: HS
authors.lastname: Lee
mesh_terms: D000558:Ambystoma mexicanum / Q000235:genetics* / Q000502:physiology*; D000671:Amputation, Surgical; D000818:Animals; D016000:Cluster Analysis; D018076:DNA, Complementary / Q000235:genetics; D003714:Denervation; D005121:Extremities / Q000294:innervation / Q000502:physiology* / Q000601:surgery; D020869:Gene Expression Profiling; D059014:High-Throughput Nucleotide Sequencing; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D058977:Molecular Sequence Annotation; D012038:Regeneration / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D014157:Transcription Factors / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D036341:Intercellular Signaling Peptides and Proteins; D014157:Transcription Factors